Studies on the "aerobic" acetyl-coenzyme A synthetase of Saccharomyces cerevisiae: purification, crystallization, and physical properties of the enzyme.

A procedure for the purification of a stable acetyl-coenzyme A synthetase (ACS) from aerobic cells of Saccharomyces cerevisiae is presented. The steps include differential centrifugation, solubilization of the bound enzyme from the crude mitochondrial fraction, ammonium sulfate fractionation, crystallization to constant specific activity from ammonium sulfate solutions followed by Bio-Gel A-1.5 m column chromatography. The resulting enzyme preparation is homogeneous as judged by chromatography on Bio-Gel columns, QAE-Sephadex A-50 anion exchange columns, analytical ultracentrifugal studies, and polyacrylamide gel electrophoresis.Sedimentation velocity runs revealed a single symmetric peak with an s_20,w value of 10.6. The molecular weight of the native enzyme, as determined by gel filtration and analytical ultracentrifugation, is 250,000 ± 500. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the single polypeptide chain is 83,000 ± 500. The purified enzyme is inhibited by palmityl-coenzyme A with a Hill interaction coefficient, n, of 2.88. These studies indicate that the ACS of aerobic S. cerevisiae is composed of three subunits of identical or nearly identical size.     

Inactivation of Kanamycin,Neomycin, and Streptomycin by Enzymes Obtained in Cells of Pseudomonas aeruginosa

Ten strains of Pseudomonas aeruginosa were disrupted and centrifuged. The supernatant fluids from centrifugation at 105,000 × g contained enzymes inactivating kanamycin, neomycin, and streptomycin in the presence of adenosine triphosphate. Kanamycin-inactivating enzyme was precipitated with ammonium sulfate at 66% of saturated concentration, and the inactivated kanamycin was shown to be kanamycin-3′-phosphate in which the C-3 hydroxyl group of 6-amino-6-deoxy-d-glucose moiety was phosphorylated. This is identical with kanamycin inactivated by Escherichia coli carrying R factor. Streptomycin-inactivating enzyme was precipitated with ammonium sulfate at 33% of saturated concentration.     

Hydrophobic chromatography of galactosyltransferase.

O-Glycosidic analogs of N-acetylglucosamine are good substrates for galactosyltransferase, and as the O-substituted group becomes more hydrophobic, the apparent K_m decreases as much as 2000-fold. l-leucine, leucine-amide, norleucine, valine, ?-amino-n-caproic acid and tyrosine-agaroses all retain galactosyltransferase in the presence of 1.25 m ammonium sulfate. The enzyme is eluted quantitatively with a five- to tenfold purification by a decreasing linear gradient of ammonium sulfate. Galactosyltransferase was not specifically bound on any of a series of ω-aminoalkylagaroses tested. A simple and highly efficient procedure for the isolation of galactosyltransferase from bovine skim milk was developed and consisted of a 40–60% ammonium sulfate precipitation of the enzyme from skim milk followed by chromatography on (1) norleucine-Sepharose, (2) UDP-hexanolamine-Sepharose, and (3) α-lactalbumin-Sepharose.     

Partial purification and properties of diacylglycerol kinase from rat liver cytosol

Diacylglycerol:ATP kinase(EC 2.3.1.-) was highly purified (more than 2000-fold) from rat liver cytosol. The specific activity of the obtained enzyme was about 1.5 μmol phosphatidate formed/mg of protein/min. The purification procedures included ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration on Sephadex G-200, and finally affinity chromatography on ATP-agarose. The activities of diacylglycerol:GTP kinase and monoacylglycerol:ATP kinase were copurified throughout the procedures, forming a single peak together with diacylglycerol: ATP kinase. Furthermore, these kinase activities showed a single peak when the highly purified enzyme was analyzed by a sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. The three kinase activities are, therefore, most likely catalyzed by a single enzyme. The kinase showed an apparent molecular weight of 121,000 on gel filtration and sedimented at 5.1 S in a sucrose gradient centrifugation. The apparent K_m values were 170 μm for ATP, 540 μm for GTP, and 3.0 μm for diacylglycerol. A number of nucleoside triphosphates and diphosphates competitively inhibited the kinase, in particular the activity utilizing GTP. Among the nucleotides tested, ADP was the most potent inhibitor (the apparent K_i:50 μm for diacylglycerol:ATP kinase and 42 μm for diacylglycerol:GTP kinase). The kinase required Mg²⁺ and deoxycholate for its activity, and the optimal pH was 8.0–8.5. No dependence on added phospholipids was observed.     

The effect of greening of sorghum leaves on the molecular weight of a complex containing 4-hydroxycinnamic Acid hydroxylase activity

During the greening of leaves of Sorghum bicolor var. Wheatland milo, the activity of 4-hydroxycinnamic (p-coumaric) acid hydroxylase in pH 6 buffered extracts was shifted from a relatively low to a high molecular weight fraction. Differences between these forms found in etiolated and green leaves were based on differential centrifugation, ammonium sulfate precipitation, and on elution patterns from Agarose A-15m. Both molecular weight forms were precipitated by protamine sulfate at pH 6, and approximately 40 to 80% of the activity of each form was associated with a 500 to 37,000g pellet when tissues were ground at pH 8 in media of either high or low osmotic concentration. Although no fraction with hydroxylase activity was ever found without any chlorogenic acid oxidase activity, the two activities frequently varied independently, and could be partially separated from each other, using the above techniques. Comparisons were made with the very small molecular weight form of 4-hydroxycinnamic acid hydroxylase characteristic of tissues of first internodes. The significance of these results in terms of possible multienzyme complexes capable of converting phenylalanine and tyrosine to cinnamic acid derivatives is discussed.     

Purification and characterization of sweet potato cytochrome c oxidase.

Sweet potato cytochrome c oxidase (EC 1.9.3.1) was purified 45-fold with respect to its specific activity, with a high recovery by solubilization of the enzyme from the submitochondrial particles with deoxycholate, diethylaminoethyl-cellulose column chromatography, and fractionation with ammonium sulfate. Impurities, if any, could be removed by sucrose density gradient centrifugation of the purified enzyme preparation, although a considerable inactivation of the enzyme took place during centrifugation. The purified enzyme contained approximately 12 nmol of heme a per milligram of protein and about 2.5% phospholipid. The cytochrome c oxidase consisted of at least five polypeptides with molecular weights of 39,000, 33,500, 26,000, 20,000, and 5700, as determined by polyacrylamide gel electrophoresis of the purified enzyme preparation in the presence of sodium dodecyl sulfate and urea. Phosphatidylcholine and phosphatidylethanolamine stimulated the activity over 3-fold. The optimal pH of the purified enzyme was 7.0 to 7.5 in the presence of phosphatidylcholine (egg yolk or soybean) and pH 6.5 in the presence of phosphatidylethanolamine.     

Activation of hormone-sensitive lipase in extracts of adipose tissue

Rat adipose tissue was homogenized in 0.154 m KCl, and the supernatant fluid, obtained after centrifugation at 15,000 g, was extracted with benzene to remove triglycerides. Most of the lipase activity in the extracted fluid was precipitated with ammonium sulfate between 15 and 40% saturation. The specific activity of the lipase in this fraction was about three times that in the benzene-extracted supernatant fluid. The specific activity of the monoglyceride esterase was increased to a lesser extent. Lipase activity in the benzene-extracted fluid and in the ammonium sulfate fraction was increased 15-45% by incubation with 0.3 mm ATP, 10 mm MgCl(2), and 0.03 mm cyclic AMP for 10 min before assay. None of these compounds alone or in combinations of two was as effective as all three together. The specific activity of the 15-40% ammonium sulfate fraction prepared from fat cells exposed to epinephrine and glucagon was greater than that from portions of the same cell pool not exposed to hormones. In addition, the already elevated lipase activity in preparations from hormone-treated cells was not enhanced by incubation with ATP, MgCl(2), and cyclic AMP. Thus, it seems probable that the lipase activity in the ammonium sulfate fractions represents, at least in part, hormone-sensitive lipase.     

Detection and Characterization of a Dehalogenating Microorganism by Terminal Restriction Fragment Length Polymorphism Fingerprinting of 16S rRNA in a Sulfidogenic, 2-Bromophenol-Utilizing Enrichment

Terminal restriction fragment length polymorphism analysis of reverse-transcribed 16S rRNA during periods of community flux was used as a tool to delineate the roles of the members of a 2-bromophenol-degrading, sulfate-reducing consortium. Starved, washed cultures were amended with 2-bromophenol plus sulfate, 2-bromophenol plus hydrogen, phenol plus sulfate, or phenol with no electron acceptor and were monitored for substrate use. In the presence of sulfate, 2-bromophenol and phenol were completely degraded. In the absence of sulfate, 2-bromophenol was dehalogenated and phenol accumulated. Direct terminal restriction fragment length polymorphism fingerprinting of the 16S rRNA in the various subcultures indicated that phylotype 2BP-48 (a Desulfovibrio-like sequence) was responsible for the dehalogenation of 2-bromophenol. A stable coculture was established which contained predominantly 2BP-48 and a second Desulfovibrio-like bacterium (designated BP212 based on terminal restriction fragment length polymorphism fingerprinting) that was capable of dehalogenating 2-bromophenol to phenol. Strain 2BP-48 in the coculture could couple reductive dehalogenation to growth with 2-bromophenol, 2,6-dibromophenol, or 2-iodophenol and lactate or formate as the electron donor. In addition to halophenols, strain 2BP-48 appears to use sulfate, sulfite, and thiosulfate as electron acceptors and is capable of simultaneous sulfidogenesis and reductive dehalogenation in the presence of sulfate.     

Purification and properties of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

Blue crab muscle (Callinectes danae) glycogen phosphorylase a was purified by adsorption of a crude extract on a starch column, elution with a dilute glycogen solution, selective precipitation with ammonium sulfate, dialysis against a solution containing ammonium sulfate and ethylenediaminetetraacetate, followed by centrifugation and chromatography on Sephadex G-25 (sp act 64.5 IU, recovery of 53.8%, and a purification factor of 189). The lyophilized preparation is stable for several months. Disc electrophoresis of the purified phosphorylase yields two protein bands, both with enzymatic activity of the a form. One of the protein bands represents about 10% of the total amount of protein present in the two bands. The molecular weight of the enzyme is 176,000 as determined by ultracentrifugation in a sucrose density gradient and 180,000 as determined by discontinuous polyacrylamide gel electrophoresis. The molecular weight found by disc electrophoresis corresponds to the main protein band. Crab muscle phosphorylase a is not associated under electrophoretic conditions in which rabbit muscle phosphorylase a shows association behavior. Subunit studies by continuous SDS-gel electrophoresis suggest that crab muscle phosphorylase a possesses only one subunit. Pyridoxal-5′-phosphate is a cofactor of the enzyme.     

Swine liver l-leucine aminopeptidase: improved purification procedure

An l-leucine aminopeptidase, having a specificity toward the substrate l-leucine amide, was purified 1084-fold from swine liver with a yield of 50.7 per cent. Purification procedure was carried out using successively centrifugation at 105 000 × g fractionation by ammonium sulfate, DEAE Sephacel chromatography and zonal ultracentrifugation.Enzyme homogeneity and purity studies were carried out by analytical ultracentrifugation and polyacrylamide gel electrophoresis.In SDS gel polyacrylamide a single band was observed. It corresponded to a 55 000 molecular weight protein.     

Cytochrome c oxidase biosynthesis and assembly in Candida utilis yeast cells. Subunit structure and molecular weight determination.

Cytochrome c oxidase was purified from mitochondria of Candida utilis yeast cells. The purification procedure involved the hypotonic incubation of mitochondria followed by washes with increasing concentrations of KCl. The membrane fragments derived from this procedure were subjected to ammonium sulfate fractionation in the presence of 2% cholate. The purified active enzyme contained 8.5–9.2 nmol heme a per mg protein and was free of other types of hemoproteins. Upon Sephadex G200 gel filtration in the presence of cholate, an apparent molecular weight of 200,000 was estimated. A single band was observed for the active enzyme upon DEAE-cellulose chromatography, sucrose density gradient centrifugation, and Sephadex G200 gel filtration.Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate resolved the enzyme into six polypeptide bands with apparent molecular weights of 49,000, 32,000, 28,000, 20,000, 13,500, and 8,000, respectively. The six components were also resolved by gel filtration on Sephadex G200, equilibrated with 0.1% sodium dodecyl sulfate, giving apparent molecular weights of 46,000, 35,000, 23,000, 19,000, 12,500, and 7,800.     

Production of Nitrous Oxide by Ammonia-Oxidizing Chemoautotrophic Microorganisms in Soil

Gas chromatographic studies showed that nitrous oxide was produced in each instance when sterilized (autoclaved) soil was incubated after treatment with ammonium sulfate and inoculation with pure cultures of ammonia-oxidizing chemoautotrophic microorganisms (strains of Nitrosomonas, Nitrosospira, and Nitrosolobus). Production of N₂O in ammonium-treated sterilized soil inoculated with Nitrosomonas europaea increased with the concentration of ammonium and the moisture content of the soil and was completely inhibited by both nitrapyrin and acetylene. Similar effects of nitrapyrin, acetylene, ammonium concentration, and soil moisture content were observed in studies of factors affecting N₂O production in nonsterile soil treated with ammonium sulfate. These observations support the conclusion that, at least under some conditions, most of the N₂O evolved from soils treated with ammonium or ammonium-producing fertilizers is generated by chemoautotrophic nitrifying microorganisms during oxidation of ammonium to nitrite.     

A simple procedure for the preparation of highly purified (sodium + potassium) adenosinetriphosphatase from the rectal salt gland of Squalus acanthias and the electric organ of Electrophorus electricus.

A simple purification procedure for the Na,K-ATPase from membranes of the rectal gland of Squalus acanthias or crude microsomal fractions from the electric organ of Electrophorus electricus is presented here. The purification procedure consists of solubilization of the Na,K-ATPase with the nonionic detergent. Lubrol WX, chromatography of the diluted Lubrol extract on aminoethyl cellulose, and ammonium sulfate fractionation (1) of the concentrated eluate from the aminoethyl cellulose column. The yields of final purified enzyme are comparable to the earlier purification (1–4) involving the expensive and cumbersome zonal centrifugation stop. The purity of the final enzyme, as attested to by specific activity and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is as great or greater than that previously reported for the enzyme purified by the procedure involving zonal centrifugation. The simplicity of the present procedure, coupled with the ready commercial availability of electric eels which are quite hardy on shipment, makes purification of the Na,K-ATPase widely available to workers in the field.     

The recovery of transfer ribonucleic acid by binding to DEAE-cellulose induced by ammonium sulfate

tRNA samples may be adsorbed quantitatively to DEAE-cellulose from 2m ammonium sulfate solution, pH 4.5. Washing with 0.3 m NaCl removes the ammonium sulfate and the tRNA is recovered in ethanol-precipitable form by elution with 1.1 m NaCl. The procedure allows convenient and efficient recovery of tRNAs from larger volumes of aqueous salt solutions. A moderate concentration of sodium chloride does not interfere with adsorption.     

Polyadenylate in the virion RNA of mouse hepatitis virus.

Mouse hepatitis (MH) virus was grown in SR-CDF1-DBT, a mouse cell line, and purified by ammonium sulfate precipitation and by density gradient centrifugation. Extraction of RNA from purified virions with 1% SDS and sedimentation analysis of the RNA revealed a major 50S component and two minor components. Treatment of virions with phenol/chloroform also produced the 50S component, although its yield was lower. MH virion RNA can bind to a poly(U)-fiberglass filter, indicating that MH virion RNA contains poly(A). A poly(A)-like fragment was isolated by digestion with ribonuclease A [EC 3.1.4.22] and T1 [EC 3.1.4.8] and by DEAE-Sephadex column chromatography. Analysis of the fragment for base composition showed it to be an adenine-rich material. Its chain length was about 90 nucleotides, as determined by ion-exchange chromatography and gel electrophoresis.     

The modulation of cell surface cAMP receptors from Dictyostelium disscoideum by ammonium sulfate

Dictyostelium discoideum cells contain a heterogeneous population of cell surface cAMP receptors with components possessing different affinities (K_d between 15 and 450 nM) and different off-rates of the cAMP-receptor complex ($\text{t}\text{1}\text{2}$ between 0.7 and 150 s). The association of cAMP to the receptor and the dissociation of the cAMP-receptor complex still occur in the presence of 3.4 M ammonium sulfate. However, these processes are strongly altered. (1) Low concentrations of ammonium sulfate (≈ 50 mM) induce an approx. 2-fold increase of the number of cAMP binding sites. The same effect is induced by millimolar concentrations of CaCl₂. Ammonium sulfate and CaCl₂ are not additive, which suggests that these salts may act via the same mechanism. (2) High concentrations of ammonium sulfate (3.4 M) induce an alteration in the proportioning of the various cAMP binding sites to the components with the highest affinity. (3) High concentrations of ammonium sulfate (3.4 M) retard the dissociation of all binding sites about 3–6-fold, thus giving rise to an increase in the affinity of all cAMP-binding components.     

Two isoenzymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leaves

Isoenzymes of glucose-6-phosphate dehydrogenase and 6-P-gluconate dehydrogenase from a 70% ammonium sulfate precipitate of spinach leaf homogenate were separated by differential solubilization in a gradient of 70-0% ammonium sulfate and analyzed by disc gel electrophoresis. Isolated whole chloroplasts contained isoenzyme 1 of both glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase 1, whereas isoenzyme 2 of each was found in the soluble cytosol fraction. Both isoenzymes of each dehydrogenase were present in about equal amounts. Glucose-6-phosphate dehydrogenase isoenzymes 1 and 2 had pH optima of 9.2 and 9.0 and K_m values of 400 and 330 μm, respectively. Molecular weights for both isoenzyme of glucose-6-phosphate dehydrogenase were very similar at about 105,000 ± 10% as estimated by sedimentation velocity measurements. For 6-phosphogluconate dehydrogenase isoenzymes 1 and 2 the pH optima were 9.0 and 9.3, respectively, the K_m values were 100 and 80 μm, and the apparent molecular weights were also nearly identical at about 110,000 ± 10%. The data support the hypothesis that leaf cells have two oxidative pentose phosphate pathways, one in the chloroplast and the other in the cytosol.     

A simplified procedure for the isolation of bacterial polypeptide elongation factor EF-Tu

Polypeptide elongation factor EF-Tu can be isolated from bacterial cell extracts in two fractionation steps. The first is ion-exchange chromatography on DEAE-Sepharose, CL-6B, and the second is gel filtration on AcA 44. The method is illustrated with extracts from Escherichia coli, Bacillus stearothermophilus, and the thermophilic bacterium PS3. The extracts were obtained from lysozyme-treated cells and were processed without high-speed centrifugation or ammonium sulfate fractionation. The procedure is simple and rapid, gives higher yields than previous methods, and is easily scaled to any size preparation. The procedure also produces fractions enriched in the other polypeptide elongation factors EF-Ts and EF-G.     

Phosphatidyl inositol: myo-Inositol exchange enzyme from rat liver: Partial purification and characterization

The enzyme which catalyzes CDP-diglyceride-independent incorporation of myo-inositol into phosphatidyl inositol was solubilized from rat liver microsomes by sodium cholate and was partially purified by ammonium sulfate fractionation and sucrose density gradient centrifugation. Addition of phospholipids during purification and assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation contained about 3.7% of the protein and 35% of the original activity of the microsomal fraction. The activity of the enzyme preparation was strongly enhanced by addition of phosphatidyl inositol. The enzyme required Mn²⁺ for activity. The K_m for myo-inositol was 4 × 10^?5m. The pH optimum was 7.4. The activity was inhibited by thiol-reactive reagents and also to some extent by inosose-2 but not by scyllitol. Phosphorus-containing acidic substances such as acidic phospholipids and nucleotides were generally inhibitory. It was found that the preparation catalyzed liberation of inositol moiety from phosphatidyl inositol in a manner dependent on the concentration of free myo-inositol and also on Mn². The K_m of this reaction for free myo-inositol was estimated to be 7 × 10^?5m. This result indicates that CDP-diglyceride-independent incorporation, which has been assumed to show inositol exchange reaction, actually represents an exchange reaction between the myo-inositol moiety of phosphatidyl inositol and free myo-inositol. Phosphatidyl choline and phosphatidyl ethanolamine did not play a role as acceptor of the exchange reaction.     

De Novo Synthesis of Isocitritase in Peanut (Arachis hypogaea L.) Cotyledons

Germination of peanut seed is accompanied by a rapid increase in isocitritase (isocitrate lyase, EC 4.1.3.1) during the first 4 days. The presence of cycloheximide (50 μg/ml) during water imbibition inhibited the increase in isocitritase activity. Actinomycin D conversely did not inhibit isocitritase activity until the second day of imbibition while RNA synthesis was inhibited. Germination of peanut seed in ¹⁴C-reconstituted amino acids followed by fractionation of a 20 to 35% ammonium sulfate preparation on a Sephadex G-200 column (57-fold purification) showed that the active enzymic fraction coincided with a large peak of radioactivity. Germination of peanut seed in 45% D₂O followed by enzyme purification and CsCl equilibrium centrifugation revealed that all the enzyme from D₂O seed had a higher density than normal isocitritase. These data indicate that isocitritase in peanut seed is synthesized de novo.